The present invention relates to the field of performing two-dimensional electrophoresis.
Electrophoresis using 2-D PAGE (Two-dimensional PolyAcrylamide Gel Electrophoresis) techniques can separate proteins in a sample according to isoelectric pH (IpH) and molecular weight. Two-Dimensional PAGE generally provides isoelectric focusing electrophoresis in one direction followed by polyacrylamide gel electrophoresis in a second direction, and can be useful in analyzing the protein composition of a sample solution such as a biological sample. For example, gene activity may be studied by analyzing various proteins important to certain cellular functions.
For example, isoelectric focusing can use Immobilized pH gradient (IPG) strips to separate proteins based on their respective IpH values. An IpH is the pH value at which a protein carries no net charge and will not migrate in an electric field. An IPG strip with zwitter ionic peptides fixed to its surface can establish an identifiable pH gradient when a voltage is applied to electrodes on opposite sides of the IPG strip. Therefore, when a protein sample is applied to an IPG strip, each protein in the sample travels through the IPG strip until it reaches its IpH value on the IPG strip.
After proteins from the sample are focused on the IPG strip, a buffer, such as a buffer including SDS (sodium dodecyl sulfate), can be applied in preparation for polyacrylamide gel electrophoresis. Sodium dodecyl sulfate is a detergent that can solubize proteins to generate a uniform negative charge. Therefore, the SDS buffer disrupts hydrophobic interactions, increases the solubility of the protein, and leaves the protein molecules negatively charged. As a result, when the proteins are exposed to a polyacrylamide gel to which an electric current is applied, the proteins travel through the polyacrylamide gel and are separated according to their molecular weight. The polyacrylamide gel is typically a flat planar gel slab supported by a cassette housing.
After completion of the isoelectric focusing in one direction and PAGE in a second direction, the polyacrylamide gel has concentrations of protein deposits that are separated by IpH in one direction and molecular weight in the other direction. In order to view the resulting protein deposits, the polyacrylamide gel can be stained, for example, by removing the gel from its cassette housing and applying a staining reagent such as a silver or ruby protein stain.
Despite producing highly resolved results, 2-D PAGE presents technical challenges that may result in low reproducibility of results. The number of manual steps involved in 2-D PAGE may require a high level of operator skill to produce reliable 2-D PAGE results. In addition to being a labor-intensive process, many technical difficulties can be caused by operator inconsistencies. For example, the isoelectric focusing steps and the polyacrylamide electrophoresis steps are often carried out using separate devices, which may introduce poor reproducibility due to operator inconsistencies. U.S. Patent Application Publication No. 2002/0100690 to Herbert (hereinafter “Herbert”), disclosure of which is hereby incorporated by reference in its entirety, proposes a method in which the IPG strip and an agarose gel slab are carried on a single cassette. Increased automation of the 2-D PAGE steps may improve reproducibility of results and decrease the need for highly skilled operators.